Abstract:

The disclosure relates to OX-2/CD200 (herein referred to as CD200)
antibodies and methods of treating autoimmune disease.

Claims:

1. A method for treating a patient who has an autoimmune or inflammatory
disease, said method comprising administering a therapeutically effective
amount of an agent which inhibits interaction between CD200 and CD200R to
said patient, wherein said autoimmune or inflammatory disease is
ameliorated.

3. The method of claim 1, wherein said agent reduces the production of
auto-antibodies.

4. The method of claim 1, wherein said agent is an anti-CD200 antibody or
antigen-binding fragment thereof.

5. The method claim 4, wherein said antibody or antibody fragment thereof
is selected from the group consisting of a polyclonal antibody, a
monoclonal antibody or antibody fragment, a recombinant antibody, a
diabody, a chimerized or chimeric antibody or antibody fragment, a
humanized antibody or antibody fragment, a deimmunized human antibody or
antibody fragment, a fully human antibody or antibody fragment, a single
chain antibody, an Fv, an Fd, an Fab, an Fab', and an F(ab').sub.2.

6. The method of claim 4, wherein said antibody is a monoclonal antibody.

7. The method of claim 1, wherein said agent has effector function.

8. The method of claim 1, wherein said agent lacks effector function.

9. The method of claim 1, wherein said agent is administered for at least
one month to said mammal.

10. The method of claim 1, wherein said agent is administered for at least
one year to said mammal.

11. The method of claim 1, wherein said agent is administered chronically
to said mammal.

12. The method of claim 1, wherein said agent is administered systemically
to said mammal.

13. The method of claim 1, wherein said agent is administered locally to
said mammal.

14. The method of claim 1, further comprising administering a second agent
or therapy.

15. The method of claim 14, wherein said second agent is administered
either sequentially or simultaneously.

16. The method of claim 1, wherein said immune response is a primary
immune response.

17. The method of claim 1, wherein said immune response is a secondary
immune response.

Description:

RELATED APPLICATIONS

[0001]This application claims the benefit of U.S. Provisional Patent
Application Ser. No. 60/962,044, filed Jul. 25, 2007, the disclosure of
which is incorporated herein by reference in its entirety.

[0003]Autoimmunity is the failure of an organism to recognize its own
constituent parts (down to the sub-molecular levels) as "self", which
results in an immune response against its own cells and tissues. Any
disease that results from such an aberrant immune response is termed an
autoimmune disease. In order to inhibit harmful immune reactions in such
instances, immunosuppressive agents such as corticosteroids and cytokine
antagonists may be administered to patients. However these general
immunosuppressives can elicit undesirable side effects including toxicity
and reduced resistance to infection. Thus alternative, and perhaps more
specific, methods of treating autoimmunity are needed.

[0004]Several immunomodulatory therapies, including antibody therapies,
have proven successful in the treatment of certain autoimmune disorders.
However there is a clinical need for additional antibody therapies for
the treatment of autoimmune disorders. Furthermore, there is a related
need for humanized or other chimeric human/mouse monoclonal antibodies.
In well publicized studies, patients administered murine anti-TNF (tumor
necrosis factor) monoclonal antibodies developed anti-murine antibody
responses to the administered antibody (Exley A. R., et al., Lancet
335:1275-1277 (1990)). This type of immune response to the treatment
regimen, commonly referred to as the human anti-mouse antibody (HAMA)
response (Mirick et al. Q J Nucl Med Mol Imaging 2004; 48: 251-7),
decreases the effectiveness of the treatment and may even render the
treatment completely ineffective. Humanized or chimeric human/mouse
monoclonal antibodies have been shown to significantly decrease the HAMA
response and to increase the therapeutic effectiveness of antibody
treatments. See, for example, LoBuglio et al., Proc. Natl. Acad. Sci. USA
86:4220-4224 (June 1989). Furthermore, antibodies in which particular
functionalities are either enhanced or reduced may find useful
applications in the clinic.

[0005]CD200, a molecule expressed on the surface of numerous cell types
including B cells, some T cells and dendritic cells and other cells,
which possesses a high degree of homology to molecules of the
immunoglobulin gene family, has previously been thought to be implicated
in immune suppression (Gorczynski et al., Transplantation 65:1106-1114
(1998)). The prior art appears to show, for example, that
CD200-expressing cells can inhibit the stimulation of Th1 cytokine
production.

[0007]In certain embodiments, said antibody or antigen-binding fragment
thereof blocks the production of auto-antibodies. In certain embodiments,
said auto-antibodies are selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA1,
IgA2, IgA, IgD, and/or IgE immunoglobulins. In certain embodiments, said
antibody or antigen-binding fragment thereof does not block the
production of auto-antibodies.

[0008]In certain embodiments, said antibody or antigen-binding fragment
thereof is an antagonistic antibody. In certain embodiments, said
antibody or antigen-binding fragment thereof is an agonistic antibody.

[0009]In certain embodiments, said antibody or antigen-binding fragment
thereof modulates expression of cytokines in said patient. In certain
embodiments, said antibody or antigen-binding fragment thereof enhances
production of a cytokine in said patient selected from the group
consisting of: IL-12, IL-10 and IL-4.

[0010]In certain embodiments, said antibody or antibody fragment thereof
is selected from the group consisting of a polyclonal antibody, a
monoclonal antibody or antibody fragment thereof, a recombinant antibody,
a diabody, a chimerized or chimeric antibody or antibody fragment
thereof, a humanized antibody or antibody fragment thereof, a deimmunized
human antibody or antibody fragment thereof, a fully human antibody or
antibody fragment thereof, a single chain antibody, an Fv, an Fd, an Fab,
an Fab', and an F(ab')2. In certain embodiments, said antibody is a
monoclonal antibody. In certain embodiments, said anti-CD200 antibody or
antibody fragment thereof is conjugated to a molecule selected from the
group consisting of a polymer and a polypeptide. In certain embodiments,
said polymer is poly(ethylene) glycol.

[0011]In certain embodiments, said antibody or antigen-binding fragment
thereof is administered for at least one month to said mammal. In certain
embodiments, said antibody or antigen-binding fragment thereof is
administered for at least one year to said mammal. In certain
embodiments, said antibody or antigen-binding fragment thereof is
administered chronically to said mammal.

[0012]In certain embodiments, said antibody or antigen-binding fragment
thereof is administered systemically to said mammal. In certain
embodiments, said antibody or antigen-binding fragment thereof is
administered locally to said mammal.

[0013]In certain embodiments, the methods of the disclosure further
comprise administering a second agent or therapy. In certain embodiments,
the second agent comprises one or more of the following characteristics:
a) regulatory activity on T cells; and b) immunomodulatory activity. In
certain embodiments, said second agent or therapy is selected from the
group consisting of an immunosuppressive agent, immunomodulatory agent,
heteroclitic peptide, antibody, antigen-binding fragment, nucleic acid,
small molecule, organometallic compound, polypeptide, aptamer,
spiegelmer, chemical, inorganic compound, metal, prodrug, and
peptidomimetic compound. In certain embodiments, the immunomodulatory or
immunosuppressive agent is a calcineurin inhibitor. In certain
embodiments, the calcineurin inhibitor is selected from tacrolimus
(FK-506) and cyclosporine A. In certain embodiments, the immunomodulatory
or immunosuppressive agent is selected from the group consisting of
adriamycin, azathiopurine, busulfan, cyclophosphamide, cyclosporine A,
Cytoxan, fludarabine, 5-fluorouracil, methotrexate, mycophenolate
mofetil, a nonsteroidal anti-inflammatory, sirolimus (rapamycin), and
tacrolimus (FK-506). In certain embodiments, the immunomodulatory or
immunosuppressive agent is an antibody selected from the group consisting
of muromonab-CD3, alemtuzumab, basiliximab, daclizumab, rituximab, IVIg
and anti-thymocyte globulin. In certain embodiments, said second agent is
administered either sequentially or simultaneously.

[0014]The invention contemplates combinations of any of the foregoing
aspects and embodiments of the invention. Other embodiments are described
in the description. All references cited herein are hereby incorporated
by reference.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1 shows anti-CD200 treatment reduces the severity of collagen
induced arthritis. DBA/1LacJ mice were administered 5 mg/kg dose of
either anti-CD200 or isotype-matched control mAb by i.p. injection from
day 1 to day 7 and day 21 to day 25 after initial BCII immunization on
day 1. Data are presented as mean±SEM.

[0016]FIG. 2 shows anti-CD200 treatment blocks the production of
anti-collagen antibody production. Serum levels and subtypes of anti-BCII
Abs were evaluated for the indicated treatment groups. Data are presented
as mean±SEM.

[0017]FIGS. 3A-3B show anti-CD200 treatment can ameliorate established
joint inflammation independent of the effect on autoantibody production.
A) DBA/1LacJ mice were administered 5 mg/kg dose of either anti-CD200 or
isotype-matched control mAb by i.p. injection on day 21 to day 30 after
initial BCII immunization on day 1. B) Serum levels and subtypes of
anti-BCII Abs were evaluated for the indicated treatment groups. Data are
presented as mean±SEM.

[0018]FIGS. 4A-4B show anti-CD200 treatment affects splenic cytokine
profiles when administered at various time points relative to collagen
immunization of DBA/1 mice. A) Spleen cells were isolated from BCII
immunized DBA/1LacJ mice, which were treated with either anti-CD200 or
isotype-matched control mAb from day 1 to day 7 and day 21 to day25 after
initial BCII immunization on day1. B) Spleen cells were isolated from
BCII immunized DBA/1LacJ mice, which were treated with either anti-CD200
or isotype-matched control mAb from day 21 to day30 after initial BCII
immunization on day1. Data are presented as mean±SEM.

[0019]FIG. 5 shows the effect of alteration of cytokine profile after
anti-CD200 treatment in an allogenic immune response, where BALB/c mice
were immunized with C57B/c spleen cells. Data are presented as
mean±SEM.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

I. Overview

[0020]Prior art, especially numerous articles by Gorczynski, has seemed to
indicate that the molecule CD200 is immunosuppressive. For example,
Gorczynski et al., Clin. Immunol. 104:256-264 (2002) teaches that in a
collagen-induced arthritis (CIA) model in mice treatment with CD200 (in a
CD200Fc form) ameliorates CIA. They state that work has shown that CD200
binds its receptor and the immunosuppressive activity is via the
receptor. The paper teaches that use of an anti-CD200 receptor antibody
that crosslinks the receptor has this same activity of ameliorating CIA,
the antibody apparently being an agonistic antibody. It is likely that
CD200, which is a member of the Ig superfamily, acts similarly to an
antibody and acts to crosslink the CD200 receptor thereby activating the
receptor which in turn results in immunosuppression.

[0021]Based on the prior art, one would expect that treatment of an animal
or patient with an anti-CD200 antibody should result in an enhanced
immune response. The logic is that antibodies to CD200 would bind the
CD200 thereby preventing CD200 from binding to its receptor. Without a
CD200:CD200 receptor interaction the CD200 receptor would not be
activated and there would be no immune suppression, thereby resulting in
enhanced inflammation or autoimmune effect. For diseases such as CIA this
would mean that treatment with anti-CD200 should exacerbate the disease.

II CD200 Antibodies

[0022]CD200 is a highly conserved type I transmembrane glycoprotein
expressed on various cell types including cells of the immune system
(e.g., T-cells, B-cells, and dendritic cells (Barclay et al., 2002 TRENDS
Immunol. 23:285-290)). The protein interacts with its receptor CD200R on
myeloid cells and sub-populations of T cells (Wright et al. J. Immunol.
2003 171: 3034-3046 and Wright et al., Immunity 2000 13:233-242); the
CD200:CD200R interaction has been thought to deliver an immunomodulatory
signal to cells and induce immunosuppression including
apoptosis-associated immune tolerance (Rosenblum et al. 2004 Blood 103:
2691-2698). Thus it has been thought that agents that modulate the
function or activity of CD200 and/or its receptor may result in enhanced
immunosuppressive effects. In addition, agents that specifically bind
CD200 (but that may or may not modulate the CD200:CD200R interaction) may
trigger downstream events that modulate the effects of CD200.

[0023]In certain aspects, the present disclosure relates to CD200
modifiers. As used herein, the term modifier includes any agent that is
capable of modulating the activity, function and/or the expression of
CD200 or its receptor. Examples include but are not limited to
polypeptides, antibodies, small molecules, aptamers, spiegelmers, locked
nucleic acid (LNA) inhibitors, peptide nucleic acid (PNA) inhibitors,
nucleic acid constructs (e.g., gene-targeting constructs, antisense
constructs, RNA interference (RNAi) constructs, etc.) and
peptidomimetics. In certain embodiments, the antibody disrupts the
interaction of CD200 and CD200R. In other embodiments, the CD200
antibodies are capable of increasing the immunosuppressive effects of
CD200 or are capable of targeting CD200-expressing cells for depletion or
elimination.

[0024]In certain aspects, the CD200 modifiers are polypeptides.
Polypeptides utilized in the present disclosure can be constructed using
different techniques which are known to those skilled in the art. In one
embodiment, the polypeptides are obtained by chemical synthesis. In other
embodiments, the polypeptides are constructed from a fragment or several
fragments. In further embodiments, the polypeptide is an anti-CD200
antibody as described herein.

[0025]As used herein, the term "antibodies" refers to complete antibodies
or antibody fragments capable of binding to CD200 or CD200R. Included are
Fab, Fv, scFv, Fab' and F(ab')2, monoclonal and polyclonal
antibodies, engineered antibodies (including chimeric, single chain,
CDR-grafted, humanized, fully human antibodies, and artificially selected
antibodies), and synthetic or semi-synthetic antibodies produced using
phage display or alternative techniques. Also included are antibodies
engineered or produced in ways to contain variant or altered constant or
Fc regions with either increased or decreased ability to bind one or more
effector cells; such variant antibodies include but are not limited to
antibodies in which the constant or Fc region contains altered
glycosylation patterns. Small fragments, such as Fv and scFv, possess
advantageous properties for diagnostic and therapeutic applications on
account of their small size and consequent superior tissue distribution.
Antibodies with engineered or variant constant or Fc regions can be
useful in modulating effector functions, such as, for example, ADCC and
CDC.

[0026]Such antibodies with engineered or variant constant or Fc regions
may be useful in instances where CD200 is expressed in normal tissue, for
example; variant anti-CD200 antibodies without effector function in these
instances may elicit the desired therapeutic response while not damaging
normal tissue. Furthermore, antibodies, variant antibodies, and fragments
thereof may be blocking (i.e., the antibodies or fragments inhibit the
interaction of CD200 and CD200R) or agonistic (i.e., the antibodies or
fragments enhance the interaction of CD200 and CD200R).

[0027]The disclosure also relates to anti-CD200 antibodies comprising
heavy and light chains as provided herein, including heavy and light
chains that are homologous or similar to the heavy and/or light chains
provided herein. "Homology" or "identity" or "similarity" refers to
sequence similarity between two peptides or between two nucleic acid
molecules. Homology and identity can each be determined by comparing a
position in each sequence which may be aligned for purposes of
comparison. When an equivalent position in the compared sequences is
occupied by the same base or amino acid, then the molecules are identical
at that position; when the equivalent site occupied by the same or a
similar amino acid residue (e.g., similar in steric and/or electronic
nature), then the molecules can be referred to as homologous (similar) at
that position. Expression as a percentage of homology/similarity or
identity refers to a function of the number of identical or similar amino
acids at positions shared by the compared sequences. The term "homology"
describes a mathematically based comparison of sequence similarities
which is used to identify genes or proteins with similar functions or
motifs. As used herein, "identity" means the percentage of identical
nucleotide or amino acid residues at corresponding positions in two or
more sequences when the sequences are aligned to maximize sequence
matching, i.e., taking into account gaps and insertions. Thus methods to
determine identity are designed to give the largest match between the
sequences tested (see Computational Molecular Biology, Lesk, A. M., ed.,
Oxford University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,
H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in
Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence
Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New
York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:
1073 (1988), Devereux, J., et al., Nucleic Acids Research 12(1): 387
(1984), BLASTP, BLASTN, FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:
403-410 (1990) and Altschul et al. Nucleic Acids Res. 25: 3389-3402
(1997)) and BLAST X (BLAST Manual, Altschul, S., et al., NCBI NLM NIH
Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410
(1990)). A sequence which is "unrelated" or "non-homologous" shares less
than 40% identity, though preferably less than 25% identity with a
sequence of the present disclosure. In comparing two sequences, the
absence of residues (amino acids or nucleic acids) or presence of extra
residues also decreases the identity and homology/similarity.

[0028]Accordingly, the disclosure relates to antibodies as described
herein without the leader sequences. Thus antibodies of the disclosure
may comprise heavy and light chains (as described herein) in which the
leader sequence is either absent or replaced by a different leader
sequence. If host cells are used to produce antibodies of the present
disclosure, appropriate leader sequences may therefore be selected
according to the particular host cell used.

[0029]Antibodies may be produced by methods well known in the art. For
example, monoclonal anti-CD200 antibodies may be generated using CD200
positive cells, CD200 polypeptide, or a fragment of CD200 polypeptide, as
an immunogen, thus raising an immune response in animals from which
antibody-producing cells and in turn monoclonal antibodies may be
isolated. The sequence of such antibodies may be determined and the
antibodies or variants thereof produced by recombinant techniques.
Recombinant techniques may be used to produce chimeric, CDR-grafted,
humanized and fully human antibodies based on the sequence of the
monoclonal antibodies as well as polypeptides capable of binding to
CD200.

[0030]Moreover, antibodies derived from recombinant libraries ("phage
antibodies") may be selected using CD200-positive cells, or polypeptides
derived therefrom, as bait to isolate the antibodies or polypeptides on
the basis of target specificity. The production and isolation of
non-human and chimeric anti-CD200 antibodies are well within the purview
of the skilled artisan.

[0031]Recombinant DNA technology is used to improve the antibodies
produced in non-human cells. Thus, chimeric antibodies may be constructed
in order to decrease the immunogenicity thereof in diagnostic or
therapeutic applications. Moreover, immunogenicity may be minimized by
humanizing the antibodies by CDR grafting and, optionally, framework
modification. See, U.S. Pat. No. 5,225,539, the contents of which are
incorporated herein by reference.

[0032]Antibodies may be obtained from animal serum or, in the case of
monoclonal antibodies or fragments thereof, produced in cell culture.
Recombinant DNA technology may be used to produce the antibodies
according to established procedure, including procedures in bacterial or
preferably mammalian cell culture. The selected cell culture system
preferably secretes the antibody product.

[0033]In another embodiment, a process for the production of an antibody
disclosed herein includes culturing a host, e.g. E. coli or a mammalian
cell, which has been transformed with a hybrid vector. The vector
includes one or more expression cassettes containing a promoter operably
linked to a first DNA sequence encoding a signal peptide linked in the
proper reading frame to a second DNA sequence encoding the antibody
protein. The antibody protein is then collected and isolated. Optionally,
the expression cassette may include a promoter operably linked to
polycistronic, for example bicistronic, DNA sequences encoding antibody
proteins each individually operably linked to a signal peptide in the
proper reading frame.

[0035]In vitro production provides relatively pure antibody preparations
and allows scale-up production to give large amounts of the desired
antibodies. Techniques for bacterial cell, yeast, plant, or mammalian
cell cultivation are known in the art and include homogeneous suspension
culture (e.g. in an airlift reactor or in a continuous stirrer reactor),
and immobilized or entrapped cell culture (e.g. in hollow fibers,
microcapsules, on agarose microbeads or ceramic cartridges).

[0036]Large quantities of the desired antibodies can also be obtained by
multiplying mammalian cells in vivo. For this purpose, hybridoma cells
producing the desired antibodies are injected into histocompatible
mammals to cause growth of antibody-producing tumors. Optionally, the
animals are primed with a hydrocarbon, especially mineral oils such as
pristane (tetramethyl-pentadecane), prior to the injection. After one to
three weeks, the antibodies are isolated from the body fluids of those
mammals. For example, hybridoma cells obtained by fusion of suitable
myeloma cells with antibody-producing spleen cells from Balb/c mice, or
transfected cells derived from hybridoma cell line Sp2/0 that produce the
desired antibodies are injected intraperitoneally into Balb/c mice
optionally pre-treated with pristane. After one to two weeks, ascitic
fluid is taken from the animals.

[0038]The cell culture supernatants are screened for the desired
antibodies, preferentially by immunofluorescent staining of
CD200-positive cells, by immunoblotting, by an enzyme immunoassay, e.g. a
sandwich assay or a dot-assay, or a radioimmunoassay.

[0039]For isolation of the antibodies, the immunoglobulins in the culture
supernatants or in the ascitic fluid may be concentrated, e.g. by
precipitation with ammonium sulfate, dialysis against hygroscopic
material such as polyethylene glycol, filtration through selective
membranes, or the like. If necessary and/or desired, the antibodies are
purified by the customary chromatography methods, for example gel
filtration, ion-exchange chromatography, chromatography over
DEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinity
chromatography with one or more surface polypeptides derived from a
CD200-positive cell line, or with Protein-A or -G.

[0040]Another embodiment provides a process for the preparation of a
bacterial cell line secreting antibodies directed against CD200 in a
suitable mammal. For example a rabbit is immunized with pooled samples
from CD200-positive tissue or cells or CD200 polypeptide or fragments
thereof. A phage display library produced from the immunized rabbit is
constructed and panned for the desired antibodies in accordance with
methods well known in the art (such as, for example, the methods
disclosed in the various references incorporated herein by reference).

[0041]Hybridoma cells secreting the monoclonal antibodies are also
disclosed. The preferred hybridoma cells are genetically stable, secrete
monoclonal antibodies described herein of the desired specificity, and
can be expanded from deep-frozen cultures by thawing and propagation in
vitro or as ascites in vivo.

[0042]In another embodiment, a process is provided for the preparation of
a hybridoma cell line secreting monoclonal antibodies against CD200. In
that process, a suitable mammal, for example a Balb/c mouse, is immunized
with one or more polypeptides or antigenic fragments of CD200 or with one
or more polypeptides or antigenic fragments derived from a CD200-positive
cell, the CD200-positive cell itself, or an antigenic carrier containing
a purified polypeptide as described. Antibody-producing cells of the
immunized mammal are grown briefly in culture or fused with cells of a
suitable myeloma cell line. The hybrid cells obtained in the fusion are
cloned, and cell clones secreting the desired antibodies are selected.
For example, spleen cells of Balb/c mice immunized with a CD200-positive
Chronic Lymphocytic Leukemia (CLL) cell line are fused with cells of the
myeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14. The obtained
hybrid cells are then screened for secretion of the desired antibodies
and positive hybridoma cells are cloned.

[0043]Preferred is a process for the preparation of a hybridoma cell line,
characterized in that Balb/c mice are immunized by injecting
subcutaneously and/or intraperitoneally between 106 and 107
cells of a CD200-positive cell line several times, e.g. four to six
times, over several months, e.g. between two and four months. Spleen
cells from the immunized mice are taken two to four days after the last
injection and fused with cells of the myeloma cell line PAI in the
presence of a fusion promoter, preferably polyethylene glycol.
Preferably, the myeloma cells are fused with a three- to twenty-fold
excess of spleen cells from the immunized mice in a solution containing
about 30% to about 50% polyethylene glycol of a molecular weight around
4000. After the fusion, the cells are expanded in suitable culture media
as described hereinbefore, supplemented with a selection medium, for
example HAT medium, at regular intervals in order to prevent normal
myeloma cells from overgrowing the desired hybridoma cells.

[0044]The antibodies and fragments thereof can be "chimeric". Chimeric
antibodies and antigen-binding fragments thereof comprise portions from
two or more different species (e.g., mouse and human). Chimeric
antibodies can be produced with mouse variable regions of desired
specificity spliced into human constant domain gene segments (for
example, U.S. Pat. No. 4,816,567). In this manner, non-human antibodies
can be modified to make them more suitable for human clinical
application.

[0045]The monoclonal antibodies of the present disclosure include
"humanized" forms of the non-human (e.g., mouse) antibodies. Humanized or
CDR-grafted mAbs are particularly useful as therapeutic agents for humans
because they are not cleared from the circulation as rapidly as mouse
antibodies and do not typically provoke an adverse immune reaction.
Generally, a humanized antibody has one or more amino acid residues
introduced into it from a non-human source. These non-human amino acid
residues are often referred to as "import" residues, which are typically
taken from an "import" variable domain. Methods of preparing humanized
antibodies are generally well known in the art. For example, humanization
can be essentially performed following the method of Winter and
co-workers (Jones et al., Nature 321:522-525 (1986); Riechmann et al.,
Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536
(1988)), by substituting rodent CDRs or CDR sequences for the
corresponding sequences of a human antibody. Also see Staelens et al.
2006 Mol Immunol 43: 1243-1257. In particular embodiments, humanized
forms of non-human (e.g., mouse) antibodies are human antibodies
(recipient antibody) in which hypervariable (CDR) region residues of the
recipient antibody are replaced by hypervariable region residues from a
non-human species (donor antibody) such as a mouse, rat, rabbit, or
non-human primate having the desired specificity, affinity, and binding
capacity. In some instances, framework region residues of the human
immunoglobulin are also replaced by corresponding non-human residues (so
called "back mutations"). In addition, phage display libraries can be
used to vary amino acids at chosen positions within the antibody
sequence. The properties of a humanized antibody are also affected by the
choice of the human framework. Furthermore, humanized and chimerized
antibodies can be modified to comprise residues that are not found in the
recipient antibody or in the donor antibody in order to further improve
antibody properties, such as, for example, affinity or effector function.

[0046]Fully human antibodies are also provided in the disclosure. The term
"human antibody" includes antibodies having variable and constant regions
(if present) derived from human germline immunoglobulin sequences. Human
antibodies can include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo).
However, the term "human antibody" does not include antibodies in which
CDR sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework sequences (i.e.,
humanized antibodies). Fully human or human antibodies may be derived
from transgenic mice carrying human antibody genes (carrying the variable
(V), diversity (D), joining (J), and constant (C) exons) or from human
cells. For example, it is now possible to produce transgenic animals
(e.g., mice) that are capable, upon immunization, of producing a full
repertoire of human antibodies in the absence of endogenous
immunoglobulin production (see, e.g., Jakobovits et al., Proc. Natl.
Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258
(1993); Bruggemann et al., Year in Immunol., 7:33 (1993); and Duchosal et
al. Nature 355:258 (1992). Transgenic mice strains can be engineered to
contain gene sequences from unrearranged human immunoglobulin genes. The
human sequences may code for both the heavy and light chains of human
antibodies and would function correctly in the mice, undergoing
rearrangement to provide a wide antibody repertoire similar to that in
humans. The transgenic mice can be immunized with the target protein
(e.g., CD200, fragments thereof, or cells expressing CD200) to create a
diverse array of specific antibodies and their encoding RNA. Nucleic
acids encoding the antibody chain components of such antibodies may then
be cloned from the animal into a display vector. Typically, separate
populations of nucleic acids encoding heavy and light chain sequences are
cloned, and the separate populations then recombined on insertion into
the vector, such that any given copy of the vector receives a random
combination of a heavy and a light chain. The vector is designed to
express antibody chains so that they can be assembled and displayed on
the outer surface of a display package containing the vector. For
example, antibody chains can be expressed as fusion proteins with a phage
coat protein from the outer surface of the phage. Thereafter, display
packages can be screened for display of antibodies binding to a target.

[0047]In addition, human antibodies can be derived from phage-display
libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech
14:309 (1996)). Synthetic phage libraries can be created which use
randomized combinations of synthetic human antibody V-regions. By
selection on antigen fully human antibodies can be made in which the
V-regions are very human-like in nature. See U.S. Pat. Nos. 6,794,132,
6,680,209, 4,634,666, and Ostberg et al. (1983), Hybridoma 2:361-367, the
contents of which are incorporated by reference.

[0049]In an alternative approach, others, including GenPharm
International, Inc., have utilized a "minilocus" approach. In the
minilocus approach, an exogenous Ig locus is mimicked through the
inclusion of pieces (individual genes) from the Ig locus. Thus, one or
more VH genes, one or more DH genes, one or more JH genes,
a mu constant region, and a second constant region (preferably a gamma
constant region) are formed into a construct for insertion into an
animal. This approach is described in U.S. Pat. No. 5,545,807 to Surani
et al. and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425,
5,661,016, 5,770,429, 5,789,650, and 5,814,318 each to Lonberg and Kay,
U.S. Pat. No. 5,591,669 to Krimpenfort and Berns, U.S. Pat. Nos.
5,612,205, 5,721,367, 5,789,215 to Berns et al., and U.S. Pat. No.
5,643,763 to Choi and Dunn, and GenPharm International. Also see U.S.
Pat. Nos. 5,569,825, 5,877,397, 6,300,129, 5,874,299, 6,255,458, and
7,041,871, the disclosures of which are hereby incorporated by reference.
See also European Patent No. 0 546 073 B1, International Patent
Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO
93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO
98/24884, the disclosures of which are hereby incorporated by reference
in their entirety. See further Taylor et al. (1992 Nucleic Acids Res.,
20: 6287), Chen et al. (1993 Int. Immunol. 5: 647), Tuaillon et al. (1993
Proc. Natl. Acad. Sci. USA. 90: 3720-4), Choi et al., (1993 Nature
Genetics 4: 117), Lonberg et al. (1994 Nature 368: 856-859), Taylor et
al. (1994 International Immunology 6: 579-591), and Tuaillon et al. (1995
J. Immunol. 154: 6453-65), Fishwild et al. (1996 Nature Biotechnology 14:
845), and Tuaillon et al. (2000 Eur. J. Immunol. 10: 2998-3005), the
disclosures of which are hereby incorporated by reference in their
entirety.

[0050]In certain embodiments, de-immunized anti-CD200 antibodies or
antigen-binding fragments thereof are provided. De-immunized antibodies
or antigen-binding fragments thereof may be modified so as to render the
antibody or antigen-binding fragment thereof non-immunogenic, or less
immunogenic, to a given species. De-immunization can be achieved by
modifying the antibody or antigen-binding fragment thereof utilizing any
of a variety of techniques known to those skilled in the art (see e.g.,
PCT Publication Nos. WO 04/108158 and WO 00/34317). For example, an
antibody or antigen-binding fragment thereof may be de-immunized by
identifying potential T cell epitopes and/or B cell epitopes within the
amino acid sequence of the antibody or antigen-binding fragment thereof
and removing one or more of the potential T cell epitopes and/or B cell
epitopes from the antibody or antigen-binding fragment thereof, for
example, using recombinant techniques. The modified antibody or
antigen-binding fragment thereof may then optionally be produced and
tested to identify antibodies or antigen-binding fragments thereof that
have retained one or more desired biological activities, such as, for
example, binding affinity, but have reduced immunogenicity. Methods for
identifying potential T cell epitopes and/or B cell epitopes may be
carried out using techniques known in the art, such as, for example,
computational methods (see e.g., PCT Publication No. WO 02/069232), in
vitro or in silico techniques, and biological assays or physical methods
(such as, for example, determination of the binding of peptides to MHC
molecules, determination of the binding of peptide:MHC complexes to the T
cell receptors from the species to receive the antibody or
antigen-binding fragment thereof, testing of the protein or peptide parts
thereof using transgenic animals with the MHC molecules of the species to
receive the antibody or antigen-binding fragment thereof, or testing with
transgenic animals reconstituted with immune system cells from the
species to receive the antibody or antigen-binding fragment thereof,
etc.). In various embodiments, the de-immunized anti-CD200 antibodies
described herein include de-immunized antigen-binding fragments, Fab, Fv,
scFv, Fab' and F(ab')2, monoclonal antibodies, murine antibodies,
engineered antibodies (such as, for example, chimeric, single chain,
CDR-grafted, humanized, fully human antibodies, and artificially selected
antibodies), synthetic antibodies and semi-synthetic antibodies.

[0051]In a further embodiment, recombinant DNA comprising an insert coding
for a heavy chain variable domain and/or for a light chain variable
domain of antibodies directed to CD200 or a CD200-positive cell line are
produced. The term DNA includes coding single stranded DNAs, double
stranded DNAs consisting of said coding DNAs and of complementary DNAs
thereto, or these complementary (single stranded) DNAs themselves.

[0052]Furthermore, DNA encoding a heavy chain variable domain and/or a
light chain variable domain of antibodies directed to CD200 or the
CD200-positive cell line can be enzymatically or chemically synthesized
DNA having the authentic DNA sequence coding for a heavy chain variable
domain and/or for the light chain variable domain, or a mutant thereof. A
mutant of the authentic DNA is a DNA encoding a heavy chain variable
domain and/or a light chain variable domain of the above-mentioned
antibodies in which one or more amino acids are deleted, inserted, or
exchanged with one or more other amino acids. Preferably said
modification(s) are outside the CDRs of the heavy chain variable domain
and/or of the light chain variable domain of the antibody in humanization
and expression optimization applications. The term mutant DNA also
embraces silent mutants wherein one or more nucleotides are replaced by
other nucleotides with the new codons coding for the same amino acid(s).
The term mutant sequence also includes a degenerate sequence. Degenerate
sequences are degenerate within the meaning of the genetic code in that
an unlimited number of nucleotides are replaced by other nucleotides
without resulting in a change of the amino acid sequence originally
encoded. Such degenerate sequences may be useful due to their different
restriction sites and/or frequency of particular codons which are
preferred by the specific host, particularly E. coli, to obtain an
optimal expression of the heavy chain murine variable domain and/or a
light chain murine variable domain.

[0053]The term mutant is intended to include a DNA mutant obtained by in
vitro mutagenesis of the authentic DNA according to methods known in the
art.

[0054]For the assembly of complete tetrameric immunoglobulin molecules and
the expression of chimeric antibodies, the recombinant DNA inserts coding
for heavy and light chain variable domains are fused with the
corresponding DNAs coding for heavy and light chain constant domains,
then transferred into appropriate host cells, for example after
incorporation into hybrid vectors.

[0055]Recombinant DNAs including an insert coding for a heavy chain murine
variable domain of an antibody directed to CD200 or a CD200-positive cell
line fused to a human constant domain IgG, for example γ1,
γ2, γ3 or γ4, in particular embodiments γ1 or
γ4, may be used. Recombinant DNAs including an insert coding for a
light chain murine variable domain of an antibody fused to a human
constant domain κ or λ, preferably κ, are also
provided.

[0056]Another embodiment pertains to recombinant DNAs coding for a
recombinant polypeptide wherein the heavy chain variable domain and the
light chain variable domain are linked by way of a spacer group,
optionally comprising a signal sequence facilitating the processing of
the antibody in the host cell and/or a DNA sequence encoding a peptide
facilitating the purification of the antibody and/or a cleavage site
and/or a peptide spacer and/or an agent. The DNA coding for an agent is
intended to be a DNA coding for the agent useful in diagnostic or
therapeutic applications. Thus, agent molecules which are toxins or
enzymes, especially enzymes capable of catalyzing the activation of
prodrugs, are particularly indicated. The DNA encoding such an agent has
the sequence of a naturally occurring enzyme or toxin encoding DNA, or a
mutant thereof, and can be prepared by methods well known in the art.

[0057]Accordingly, the monoclonal antibodies or antigen-binding fragments
of the disclosure can be naked antibodies or antigen-binding fragments
that are not conjugated to other agents, for example, a therapeutic agent
or detectable label. Alternatively, the monoclonal antibody or
antigen-binding fragment can be conjugated to an agent such as, for
example, a cytotoxic agent, a small molecule, a hormone, an enzyme, a
growth factor, a cytokine, a ribozyme, a peptidomimetic, a chemical, a
prodrug, a nucleic acid molecule including coding sequences (such as
antisense, RNAi, gene-targeting constructs, etc.), or a detectable label
(e.g., an NMR or X-ray contrasting agent, fluorescent molecule, etc.). In
certain embodiments, an anti-CD200 polypeptide or antigen-binding
fragment (e.g., Fab, Fv, single-chain scFv, Fab' and F(ab')2) is
linked to a molecule that increases the half-life of the polypeptide or
antigen-binding fragment. Molecules that may be linked to said anti-CD200
polypeptide or antigen-binding fragment include but are not limited to
serum proteins including albumin, polypeptides, other proteins or protein
domains, and PEG.

[0060]In certain embodiments, an anti-CD200 antibody may be a blocking or
agonistic. As used herein, a blocking antibody is one that blocks the
interaction between CD200 and CD200R. An agonistic antibody is one that
enhances the interaction between CD200 and CD200R. Thus in certain
embodiments, an anti-CD200 antibody is either a blocking or agonistic
murine, chimeric, humanized, human or de-immunized antibody.

[0061]The CD200 antibodies and polypeptides and/or antibodies utilized in
the present disclosure are especially indicated for diagnostic and
therapeutic applications as described herein. Accordingly CD200
antibodies and anti-CD200 antibodies and variants thereof may be used in
therapies, including combination therapies, in the diagnosis and
prognosis of disease, as well as in the monitoring of disease
progression.

[0062]In the therapeutic embodiments of the present disclosure, bispecific
antibodies are contemplated. Bispecific antibodies are monoclonal,
preferably human or humanized, antibodies that have binding specificities
for at least two different antigens. In the present case, one of the
binding specificities is for the CD200 antigen on a cell (such as, e.g.,
an immune cell), the other one is for any other antigen, and preferably
for a cell-surface protein or receptor or receptor subunit.

[0063]Methods for making bispecific antibodies are within the purview of
those skilled in the art. Traditionally, the recombinant production of
bispecific antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have different
specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Antibody
variable domains with the desired binding specificities (antibody-antigen
combining sites) can be fused to immunoglobulin constant domain
sequences. The fusion preferably is with an immunoglobulin heavy-chain
constant domain, including at least part of the hinge, CH2, and CH3
regions. DNAs encoding the immunoglobulin heavy-chain fusions and, if
desired, the immunoglobulin light chain, are inserted into separate
expression vectors, and are co-transfected into a suitable host organism.
For further details of illustrative currently known methods for
generating bispecific antibodies see, for example, Suresh et al., Methods
in Enzymology, 121:210 (1986); WO 96/27011; Brennan et al., Science
229:81 (1985); Shalaby et al., J. Exp. Med. 175:217-225 (1992); Kostelny
et al., J. Immunol. 148(5):1547-1553 (1992); Hollinger et al., Proc.
Natl. Acad. Sci. USA 90:6444-6448 (1993); Gruber et al., J. Immunol.
152:5368 (1994); and Tutt et al., J. Immunol. 147:60 (1991). Bispecific
antibodies also include cross-linked or heteroconjugate antibodies.
Heteroconjugate antibodies may be made using any convenient cross-linking
methods. Suitable cross-linking agents are well known in the art, and are
disclosed in U.S. Pat. No. 4,676,980, along with a number of
cross-linking techniques.

[0064]Various techniques for making and isolating bispecific antibody
fragments directly from recombinant cell culture have also been
described. For example, bispecific antibodies have been produced using
leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).
The leucine zipper peptides from the Fos and Jun proteins may be linked
to the Fab' portions of two different antibodies by gene fusion. The
antibody homodimers may be reduced at the hinge region to form monomers
and then re-oxidized to form the antibody heterodimers. This method can
also be utilized for the production of antibody homodimers. The "diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for making
bispecific antibody fragments. The fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) by a linker which is too short to allow pairing between the two
domains on the same chain. Accordingly, the VH and VL domains
of one fragment are forced to pair with the complementary VL and
VH domains of another fragment, thereby forming two antigen-binding
sites. Another strategy for making bispecific antibody fragments by the
use of single-chain Fv (scFv) dimers has also been reported. See Gruber
et al., J. Immunol., 152:5368 (1994). Alternatively, the antibodies can
be "linear antibodies" as described in Zapata et al. Protein Eng.
8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of
tandem Fd segments (VH--CH1--VH--CH1) which form a
pair of antigen binding regions. Linear antibodies can be bispecific or
monospecific.

III. Methods of Treating Patients with Autoimmune Disorders

[0065]In certain aspects, the disclosure relates to treating patients with
autoimmune disorders with a therapy comprising an anti-CD200 antibody or
antigen-binding fragment thereof. In certain aspects, the disclosure
relates to treating patients with an unwanted immune response with a
therapy comprising an anti-CD200 antibody or antigen-binding fragment
thereof. The antibody may be antagonistic, agonistic or a non-blocking
antibody and may be a murine, chimeric, humanized, human or de-immunized
anti-CD200 antibody. Thus, methods of treating patients with autoimmune
disorders or an unwanted immune response may comprise any of the CD200
antibodies as set forth in the present disclosure.

[0066]In certain embodiments, anti-CD200 antibodies may be used for
depleting any type of cell that expresses CD200 on its surface, including
for example, immune cells such as T-cells, B-cells, and dendritic cells.
In one embodiment, anti-CD200 antibodies may be useful for targeted
destruction of immune cells involved in an unwanted immune response.

[0067]In certain aspects, the disclosure relates to treating patients with
autoimmune disorders or an unwanted immune response with a therapy
comprising an anti-CD200 antibody or antigen-binding fragment thereof
that blocks the production of auto-antibodies. In certain embodiments,
said auto-antibodies are selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA1,
IgA2, IgA, IgD, and/or IgE immunoglobulins. In certain embodiments, said
antibody may be any antibody or antigen-binding fragment thereof of the
application. In certain embodiments, an antibody or antigen-binding
fragment thereof of the application does not block the production of
auto-antibodies.

[0068]In certain aspects, the disclosure relates to treating patients with
autoimmune disorders or an unwanted immune response with a therapy
comprising an anti-CD200 antibody or antigen-binding fragment thereof
that modulates expression of cytokines in said patient. In certain
embodiments, said antibody or antigen-binding fragment thereof enhances
production of a cytokine in said patient selected from the group
consisting of: IL-12, IL-10 and IL-4. In certain embodiments, said
antibody or antigen-binding fragment thereof modulates production of a
cytokine in said patient selected from the group consisting of: IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,
IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, and
IL-33. In certain embodiments, said antibody may be any antibody or
antigen-binding fragment thereof of the application.

[0070]Therapies comprising CD200 antibodies may be administered to
patients in combination therapies. Accordingly, targeted killing of
certain populations of immune cells for treating or preventing autoimmune
disorders, enhancing or extending transplant survival, treating or
preventing allergies, or treating or preventing inflammatory disorders,
may be administered as part of a combination therapy. For example, a
patient receiving a first therapy comprising a CD200 antibody (e.g., an
anti-CD200 antibody described herein) may also be given a second therapy.
The CD200 antibody may be given simultaneously with the second therapy.
Alternatively, the CD200 antibody may be given prior to or following the
second therapy. Second therapies include but are not limited to
anti-inflammatory agents, immunosuppressive agents, and/or anti-infective
agents.

[0071]Combination therapies of the present disclosure include, for
example, a CD200 antibody as described herein administered concurrently
or sequentially in series with steroids, anti-malarials, aspirin,
non-steroidal anti-inflammatory drugs, immunosuppressants, or cytotoxic
drugs. Included are corticosteroids (e.g. prednisone, dexamethasone, and
prednisolone), methotrexate, methylprednisolone, macrolide
immunosuppressants (e.g. sirolimus and tacrolimus), mitotic inhibitors
(e.g. azathioprine, cyclophosphamide, and methotrexate), fungal
metabolites that inhibit the activity of T lymphocytes (e.g.
cyclosporine), mycophenolate mofetil, glatiramer acetate, and cytotoxic
and DNA-damaging agents (e.g. chlorambucil). For autoimmune disorders
anti-CD200 therapy may be combined with antibody treatments including
daclizumab, a genetically engineered human IgG1 monoclonal antibody that
binds specifically to the α-chain of the interleukin-2 receptor, as
well as various other antibodies targeting immune cells or other cells.
Such combination therapies may be useful in the treatment of type 1
diabetes, rheumatoid arthritis, lupus, and idiopathic thrombocytopenic
purpura, and other autoimmune indications. The disclosure also relates to
therapies for autoimmune disorders and for transplant patients comprising
a CD200 antibody (such as, for example, the antibodies and variants
thereof described in the present disclosure) conjugated to one or more
agent.

IV. Modes of Administration and Formulations

[0072]The route of antibody administration of the antibodies of the
present disclosure (whether the pure antibody, a labeled antibody, an
antibody fused to a toxin, etc.) is in accord with known methods, e.g.,
injection or infusion by intravenous, intraperitoneal, intracerebral,
intramuscular, subcutaneous, intraocular, intraarterial, intrathecal,
inhalation or intralesional routes, or by sustained release systems. The
antibody is preferably administered continuously by infusion or by bolus
injection. One may administer the antibodies in a local or systemic
manner.

[0073]The present antibodies may be prepared in a mixture with a
pharmaceutically acceptable carrier. Techniques for formulation and
administration of the compounds of the instant application may be found
in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton,
Pa., latest edition. This therapeutic composition can be administered
intravenously or through the nose or lung, preferably as a liquid or
powder aerosol (lyophilized). The composition may also be administered
parenterally or subcutaneously as desired. When administered
systemically, the therapeutic composition should be sterile,
substantially pyrogen-free and in a parenterally acceptable solution
having due regard for pH, isotonicity, and stability. For example, a
pharmaceutical preparation is substantially free of pyrogenic materials
so as to be suitable for administration as a human therapeutic. These
conditions are known to those skilled in the art.

[0074]In certain embodiments, any antibody or antigen-binding fragment
thereof of the application is administered acutely to said mammal. In
certain embodiments, said antibody or antigen-binding fragment thereof is
administered for at least one month to said mammal. In certain
embodiments, said antibody or antigen-binding fragment thereof is
administered for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months to
said mammal. In certain embodiments, said antibody or antigen-binding
fragment thereof is administered for at least one year to said mammal. In
certain embodiments, said antibody or antigen-binding fragment thereof is
administered for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 years to said
mammal. In certain embodiments, said antibody or antigen-binding fragment
thereof is administered chronically to said mammal, i.e., recurrently for
at least 14 days, 28 days, 3 months, 6 months, 1 year, 5 years, or
longer. In certain embodiments, said antibody or antigen-binding fragment
thereof is administered to said mammal for the remainder of its life.

[0075]Pharmaceutical compositions suitable for use include compositions
wherein one or more of the present antibodies are contained in an amount
effective to achieve their intended purpose. More specifically, a
therapeutically effective amount means an amount of antibody effective to
prevent, alleviate or ameliorate symptoms of disease or prolong the
survival of the subject being treated. Determination of a therapeutically
effective amount is well within the capability of those skilled in the
art, especially in light of the detailed disclosure provided herein.
Therapeutically effective dosages may be determined by using in vitro and
in vivo methods.

EXEMPLIFICATION

Example 1

Materials and Methods

Induction and Evaluation of Collagen Induced Arthritis (CIA)

Preparation of Reagents:

[0076]1. Preparation of BCII: (Bovine type II Collagen from Elastin
Products).BCII is reconstituted by stirring overnight in cold room in
0.01 M Acetic Acid at a concentration of 4 mg/mL.

2. Preparation of Complete Freund's Adjuvant (CFA) H37Ra (Difco):

[0077]10 mg Mycobacterium tuberculosis is added to 10 ml Complete Freund's
Adjuvant which contains 10 mg Mycobacterium tuberculosis so that the
final concentration for Mycobacterium tuberculosis is 2 mg/mL. CFA is
stirred overnight at 4° C. overnight.3. Preparation of emulsion
(4° C.):1:1 ratio of 4 mg/mL BCII and CFA with Mycobacterium
tuberculosis (2 mg/mL) 100 μL emulsion=200 μg BCII+100 μg
CFA-M.T H37Ra4. Intradermal injection:Inject 150 μL of emulsion
intradermally at the base of the mouse tail.Mice will be re-immunized 21
days after the first immunization following the identical protocol.

R/SW--redness and swelling [0085]2. The degree of swelling was visually
examined and measured with a caliper at the following time points:
[0086]Time: 1) before immunization. [0087]2) once/day starting from day
21 to day 42.A Total Arthritic Score was calculated by adding the
measurements for each paw, elbow and knee (total of 8 measurements per
mouse thus yielding a maximal score of 24).

Serum Collection Time:

[0087][0088]1. Before immunization [0089]2. day 14 and day 28 for
prevention treatment group, day 31 for therapeutic treatment group.
[0090]3. day 42 after first immunization [0091]4. Serum anti-collagen
antibodies (B cell response) were measured at the times indicated in
FIGS. 2 and 3B. [0092]5. Spleen cells for cytokine measurement were taken
when the animals were sacrificed.Histological Examination: Day 42 after
First Immunization

[0093]1. Collect animal spleen in plain HBSS or PBS on ice;2. Homogenize
the spleen to collect splenocytes in 5-10 mL of plain HBSS or PBS, spin
down the cells by 1,250 rpm×5 min, rm. tp.;3. Discard supernatant,
re-suspend the cell pellet by vortex, add 5-10 mL of ACK cell lysis
buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA
2H2O) on to the cells for 3 min. at rm. tp.;4. Add FACS
Washing/Staining Buffer (2% FBS/HBSS+0.02% Sodium Azide) fill to the top
of the tube and spin down the cells by 1,250 rpm×5 min, rm. tp.;5.
Repeat wash twice;6. Count the cells and then distribute
1.0×106 cells/50 μL/well in to the 96 well U-bottom
plate;7. Add antibodies (0.1 μg/well=1.0×106 cells) into
the wells according to the staining plan, if both antibodies for the
double staining are for the cell surface markers these can be added
together in this step, for 30-60 min. 4° C. in dark;8. Repeat wash
3 times using FACS washing/staining buffer, 250 μL/well;9. Add CytoFix
buffer (BD Pharmingen Kit) 250 μL/well for 30 min. 4° C. in
dark;10. Wash wells with CytoPerm/CytoWash 3 times;11. Re-suspend cells
with 50 μL/well of CytoPerm/CytoWash buffer, add antibodies against
cytokines (0.1 μg/well=1.0×106 cells), incubate for 30-60
min. 4° C. in dark;12. Repeat wash with FACS washing buffer twice,
see above #8, wash plate once with plain PBS;Re-suspend wells to add 250
μL/well of plain PBS, transfer the cells into the FACS tube. The
samples are now ready for running the FACS.

Example 2

Evaluation of Anti-CD200 on Arthritis Animal Model

[0094]Administration of anti-CD200 antibody was performed in mice to test:
1) whether administration of anti-CD200 antibody prevents the development
of arthritis and 2) whether administration of anti-CD200 antibody reduces
the severity of existing arthritis. A collagen induced arthritis (CIA)
mouse model was used (mouse strain: DBA/1LacJ from Jackson Labs, male, 8
to 12 weeks old).

[0095]The anti-CD200 mAb used was OX90mG2a, a chimeric antibody derived
from OX90, a rat anti-mouse CD200 mAb obtained as a hybridoma from the
European Collection of Cell Cultures (ECACC No. 03062502; see Hoek et
al., Science 290:1768-1771 (2000)). The rat antibody was genetically
modified to contain the rat heavy chain variable regions fused to a
murine IgG2a constant region and the rat light chain variable region
fused to a murine kappa constant region. An isotype matched control mAb,
r12B4 was used as a control.

[0096]A) Prevention of Arthritis

[0097]Ten DBA/1LacJ mice were administered a 5 mg/kg dose of either
anti-CD200 or isotype-matched control mAb by i.p. injection from day 1 to
day 7 and day 21 to day 25 after initial BCII immunization on day 1. An
additional 10 mice were treated again at day 21 to day 25 and were
terminated at day 42. Mice were bled at day 14 to measure antibody
response.

[0098]As seen in FIG. 1, anti-CD200 treatment reduces the severity of
collagen induced arthritis (FIG. 1). Anti-CD200 treatment also inhibits
the production of anti-collagen antibody production (FIG. 2). Serum
levels and subtypes of anti-BCII Abs were evaluated for the indicated
treatment groups. DBA/1LacJ mice were i.p. injected with either
anti-CD200 or isotype-matched control mAb from day 1 to day 7 and day 21
to day 25 and bled at pre-immunization, day 14, day 28 and day 42 to day
45 after initial BCII immunization on day 1.

[0099]B) Amelioration of Established Arthritis

[0100]DBA/1LacJ mice were administered a 5 mg/kg dose of either anti-CD200
or isotype-matched control mAb by i.p. injection on day 21 to day 30
after initial BCII immunization on day 1. Serum levels and subtypes of
anti-BCII Abs were evaluated for the indicated treatment groups. DBA/1
LacJ mice were i.p. injected with either anti-CD200 or isotype-matched
control mAb from day 21 to day 30 and bled at pre-immunization, day 14,
day 28 and day 42 to day 45 after initial BCII immunization on day 1. As
shown in FIGS. 3A-B, anti-CD200 treatment can ameliorate established
joint inflammation independently of the effect on autoantibody
production.

[0101]Anti-CD200 treatment affects splenic cytokine profiles when
administered at various time points relative to collagen immunization of
DBA/1 mice (FIGS. 4A-4B). Spleen cells were isolated from BCII immunized
DBA/1LacJ mice, which were treated with either anti-CD200 or
isotype-matched control mAb from day 1 to day 7 and day 21 to day25 after
initial BCII immunization on day 1. The percentage of IL-4, IL-10,
TNF-α and INF-γ producing cells were analyzed by
intracellular staining with anti-IL-4, anti-IL-10, anti-TNF-α,
anti-INF-γ or with isotype-matched control IgG1 Ab. Spleen cells
were isolated from BCII immunized DBA/1LadJ mice, which were treated with
either anti-CD200 or isotype-matched control mAb from day 21 to day30
after initial BCII immunization on day 1. The percentage of IL-4, IL-10,
TNF-α and INF-γ producing cells were analyzed by
intracellular staining with anti-IL-4, anti-IL-10, anti-TNF-α,
anti-INF-γ or with isotype-matched control IgG1 Ab.

[0102]The effect of alteration of cytokine profile after anti-CD200
treatment was further demonstrated in an allogenic immune response, where
BALB/c mice were immunized with C57B/c spleen cells (FIG. 5).

[0103]It will be understood that various modifications may be made to the
embodiments disclosed herein. For example, as those skilled in the art
will appreciate, the specific sequences described herein can be altered
slightly without necessarily adversely affecting the functionality of the
polypeptide, antibody or antibody fragment used in binding OX-2/CD200.
For instance, substitutions of single or multiple amino acids in the
antibody sequence can frequently be made without destroying the
functionality of the antibody or fragment. Thus, it should be understood
that polypeptides or antibodies having a degree of identity greater than
70% to the specific antibodies described herein are within the scope of
this disclosure. In particularly useful embodiments, antibodies having an
identity greater than about 80% to the specific antibodies described
herein are contemplated. In other useful embodiments, antibodies having
an identity greater than about 90% to the specific antibodies described
herein are contemplated. Therefore, the above description should not be
construed as limiting, but merely as exemplifications of preferred
embodiments. Those skilled in the art will envision other modifications
within the scope and spirit of this disclosure.